Immunotherapy of cancer using genetically engineered gd2-specific t cells

ABSTRACT

The present invention concerns immunotherapy for cancers having cells that comprise the ganglioside GD2 antigen. In specific embodiment, T cells having a chimeric receptor that targets GD2 is employed. In particular cases, the chimeric receptor comprises antibody, cytoplasmic signaling domain from the T cell receptor, and/or costimulatory molecule(s).

This application is a divisional of U.S. patent application Ser. No.13/820,931 filed Apr. 3, 2013 which is a national phase applicationunder 35 U.S.C. §371 that claims priority to International ApplicationNo. PCT/US2011/050780 filed Sep. 8, 2011, which claims priority to U.S.Provisional Application Ser. No. 61/380,761 filed Sep. 8, 2010, all ofwhich are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under PO1 CA94237awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present invention generally concerns the fields of cell biology,molecular biology, and medicine. In particular, the field of theinvention concerns immunotherapy of cancer.

BACKGROUND OF THE INVENTION

The rising incidence of cutaneous melanoma and the failure tosignificantly improve outcomes in metastatic disease have led toincreasing interest in immunotherapeutic approaches, because these canbe remarkably effective (Jemal et al., 2008; Bajetta et al., 2002;Rosenberg et al., 2008). Several investigators have focused on targetingtumor-associated antigens that fall into the cancer testis antigengroup, including MAGE, BAGE, GAGE, and NY-ESO-1, or the melanocytedifferentiation protein group, including gp100, Melan-A/MART-1, andtyrosinase, which are widely present on melanoma cells. These studieshave used cytotoxic T cell lines (Mackensen et al., 2006; Butler et al.,2007), clones with native (Rosenberg et al., 2004) or transgenic αβ Tcell receptors (Morgan et al., 2006) specific for cancer testisantigen-derived peptides that are recognized in association with humanleukocyte antigen (HLA) class I antigens on the tumor cell surface. Itis clear, however, that the heterogeneity of protein antigen expressionand presentation in melanoma is a characteristic that helps limit theproportion of patients who are able to respond to such targetedstrategies (Ohnmacht and Marincola, 2000). One means of increasing theeffectiveness of targeted T cell therapy of melanoma, therefore, may beto use artificial chimeric receptors derived, for example, from theantigen binding domain of a monoclonal antibody (Pule et al., 2003).When coupled to appropriate intracellular signaling domains, T cellsexpressing these chimeric antigen receptors (CAR) can kill tumor celltargets (Haynes et al., 2002). They have the advantage of acting in aMHC unrestricted manner, allowing them to target tumor cells in whichantigen processing or presentation pathways are disrupted. Moreover,they can be directed to nonpeptide antigens on the cell surface,broadening the range of target structures that can be recognized onmalignant cells. Hence, CAR-expressing T cells could complement MHCrestricted cytotoxic T cells, and increase the overall effectiveness ofthis cellular immunotherapy.

Many melanoma cells express a range of gangliosides, including GD2, GM2,GM3, and GD3, that may be a good choice of target for CAR-T cells,because their expression is highly tissue-restricted (Yun et al., 2000;Livingston, 1998). Although these carbohydrate antigens are expressed byboth normal melanocytes and melanoma cells, expression is significantlyup-regulated after malignant transformation of melanocytes (Tsuchida etal., 1989; Albino et al., 1992), and is associated with changes in theproliferation, migration, and metastatic potential of the tumor cells(Ravindranath et al., 2008). Moreover, natural or vaccine-inducedantibodies to gangliosides in melanoma patients have been correlatedwith improved disease relapse-free survival (Livingston et al., 2008;Ragupathi et al., 2003).

BRIEF SUMMARY OF THE INVENTION

The present invention concerns methods and compositions for thetreatment of cancer, including treatment of cancer employingimmunotherapy. In particular cases, the immunotherapy includes Tlymphocytes engineered to target certain cancers. Although any cancersmay be targeted using the inventive therapy (including brain, breast,pancreatic, liver, kidney, lung, spleen, gall bladder, anal, testicular,ovarian, cervical, skin, bone, blood, or colon, for example), inparticular cases the cancer is melanoma or lung cancer, including nonsmall cell lung cancer. In specific embodiments, the cancer beingtreated has cancer cells with GD2 as an antigen on the surface of thecancer cells. In particular cases, the cytotoxic T lymphocytes (CTLs)employed to target GD2 on the surface of cancer cells comprise areceptor for GD2 and, in specific cases, the receptor on the CTLs ischimeric, non-natural and engineered at least in part by the hand ofman. In particular cases, the engineered chimeric antigen receptor (CAR)has one, two, three, four, or more components, and in some embodimentsthe one or more components facilitate targeting or binding of the Tlymphocyte to the GD2 antigen-comprising cancer cell, although in somecases one or more components are useful to promote T cell growth andmaturity.

In certain embodiments, the present invention includes T lymphocytesengineered to comprise a chimeric receptor having an antibody for GD2,part or all of a cytoplasmic signaling domain, and/or part or all of oneor more costimulatory molecules, for example endodomains ofcostimulatory molecules. In specific embodiments, the antibody for GD2is a single-chain variable fragment (scFv), although in certain aspectsthe antibody is directed at other target antigens on the cell surface,such as HER2 or CD19, for example. In certain embodiments, a cytoplasmicsignaling domain, such as those derived from the T cell receptorζ-chain, is employed as at least part of the chimeric receptor in orderto produce stimulatory signals for T lymphocyte proliferation andeffector function following engagement of the chimeric receptor with thetarget antigen. Examples would include, but are not limited to,endodmains from co-stimulatory molecules such as CD28, 4-1BB, and OX40or the signalling components of cytokine receptors such as IL7 and IL15.In particular embodiments, costimulatory molecules are employed toenhance the activation, proliferation, and cytotoxicity of T cellsproduced by the CAR after antigen engagement. In specific embodiments,the costimulatory molecules are CD28, OX40, and 4-1BB and cytokine andthe cytokine receptors are IL7 and IL15.

Genetic engineering of human T lymphocytes to express tumor-directedchimeric antigen receptors (CAR) can produce antitumor effector cellsthat bypass tumor immune escape mechanisms that are due to abnormalitiesin protein-antigen processing and presentation. Moreover, thesetransgenic receptors can be directed to tumor-associated antigens thatare not protein-derived, such as the ganglioside GD2, which is expressedin a high proportion of melanoma cells.

In certain embodiments, the present invention provides chimeric T cellsspecific for the ganglioside GD2 by joining an extracellularantigen-binding domain derived from the GD2-specific antibody sc14.G2ato cytoplasmic signaling domains derived from the T-cell receptorζ-chain, with the endodomains of the exemplary costimulatory moleculesCD28 and OX40, for examples. This CAR was expressed in human T cells andthe targeting of GD2-positive melanoma tumors was assessed in vitro andin a murine xenograft, for example.

As described herein, upon coincubation with GD2-expressing melanomacells, CAR-GD2 T lymphocytes incorporating the CD28 and OX40 endodomainssecreted significant levels of cytokines in a pattern comparable withthe cytokine response obtained by engagement of the native CD3 receptor.These CAR-T cells had antimelanoma activity in vitro and in an exemplaryxenograft model, increasing the survival of tumor-bearing animals. Thus,redirecting human T lymphocytes to the tumor-associated ganglioside GD2generates effector cells with antimelanoma activity that is useful insubjects with disease.

In some embodiments, there is a method of targeting a cancer cell havinga GD2 antigen, comprising the steps of providing to the cell a cytotoxicT lymphocyte with a chimeric receptor that recognizes the GD2 antigen.In specific embodiments, the cancer cell is in vitro or in vivo. Incertain embodiments, the chimeric receptor comprises antibody that bindsGD2, such as a scFv antibody, for example the 14g2a scFv antibody.

In particular embodiments, the chimeric receptor comprises the effectordomain of the T-cell receptor zeta chain or related signal transductionendodomains derived from the T cell receptor. In specific cases, thechimeric receptor comprises one or more costimulatory molecules, such asCD28, OX40, 4-1BB, or a combination thereof, for example. In specificembodiments, the cancer cell is in an individual with melanoma or nonsmall cell lung cancer.

In one embodiment of the present invention, there is a method oftreating melanoma or non small cell lung cancer in an individual,comprising the steps of administering to an individual cytotoxic Tlymphocytes having a chimeric receptor that recognizes a GD2 antigen onthe surface of cancer cells. In certain embodiments, the chimericreceptor comprises antibody that binds GD2, for example a scFv antibody,such as the 14g2a scFv antibody, as one instance.

In particular cases the chimeric receptor comprises the effector domainof the T-cell receptor zeta chain. In a specific embodiment, thechimeric receptor comprises one or more costimulatory molecules, such asCD28, OX40, 4-1BB, or a combination thereof, for example. In specificaspects, the individual has had and/or is having an additional cancertherapy for the respective melanoma or non small cell lung cancer.

The foregoing has outlined some of the features and technical advantagesof the present invention in order that the detailed description of theinvention that follows may be better understood. Additional features andadvantages of the invention will be described hereinafter which form thesubject of the claims of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression of GD2 antigen in human melanoma cell lines. Theexpression of GD2 was evaluated by FACS analysis in 11 melanoma celllines. Six (45%) and three (27%) tumor cell lines showed GD2 expressionat intermediate/high (++/+++) or low levels (+), respectively. In twotumor cell lines (18%) GD2 was undetectable. A GD2-normal skinfibroblast line was used as a negative control for GD2 expression. Openhistograms, isotype control of the GD2 staining (grey histograms).

FIGS. 2A and 2B demonstrate that T lymphocytes can be geneticallymodified to express CARs targeting GD2. Activated T lymphocytes weregenetically modified to express CAR-GD2. FIG. 2A shows expression ofCAR-GD2 as assessed by FACS analysis using a specific 14.g2aanti-idiotype antibody (1A7). Graph is a representative expression ofCAR-GD2 from four different transduced T cell lines. FIG. 2B shows thatboth CD4-positive and CD8-positive T lymphocytes expressed the CAR-GD2after gene transfer.

FIG. 3 demonstrates lymphocytes redirected to express CAR-GD2 killGD2-positive melanoma cell lines. A ⁵¹Cr release assay was used toevaluate the cytotoxic activity of T lymphocytes expressing CAR-GD2 andnontransduced (NT) T cells. Target cells were melanoma lines with absentGD2 (4405 M) or low (CBL), intermediate (SEMMA), or high (P1143) GD2expression. Left and right graphs, mean and SD of ⁵¹Cr release from fourT cell lines after 6 and 18 h incubation, respectively.

FIG. 4 shows T lymphocytes expressing GD2-CAR produce Th1 and Th2cytokines in response to GD2-positive melanoma cell lines. T lymphocytestransduced with CAR-GD2 or nontransduced (NT) T cells were cocultured(ratio T lymphocytes:tumor cells of 20:1) with four different melanomacell lines either negative for GD2 (4405 M) or expressing dim (CBL),intermediate (SEMMA), or high (P1143) levels of GD2. Culture supernatantwas collected 24 h later and the production of IL-2, IL-5, IFN-γ, andTNF-α measured using a CBA assay. Neither IL-4 nor IL-10 was detected inthe 24-h supernatants. The results of four experiments are presented.

FIGS. 5A and 5B provide T-lymphocytes redirected to express CAR-GD2eliminate GD2-positive melanoma cell lines in vitro. To evaluate thecapacity of T lymphocytes expressing CAR-GD2 to eliminate melanomacells, nontransduced (NT) or CAR-GD2 transduced T lymphocytes werecultured with melanoma cell lines that were GD2-negative (4405 M) orexpressed dim (CBL), intermediate (SEMMA), or high (P1143) levels of thetarget antigen. T lymphocytes and melanoma cell lines were plated at20:1 ratio and cultured for 5 d without adding IL-2 to the culture.Residual melanoma cells were enumerated by FACS analysis. In FIG. 5A,there are mean and SD of surviving cells expressing GFP for four T celllines. In FIG. 5B, there is phenotypic analysis of cocultureexperiments.

FIGS. 6A and 6B demonstrates that CAR-GD2 T lymphocytes control tumorgrowth in vivo. SCID mice were infused i.v. with 2×10⁶ melanoma cellsfrom the cell lines 4405 M (0% GD2 positive) or P1143 (95% GD2 positive)labeled with FFLuc gene. Tumor growth and engraftment was monitoredusing an in vivo imaging system (Xenogen-IVIS Imaging System). Four and21 d after tumor infusion, mice were treated with T lymphocytes CAR-GD2or nontransduced (NT) T cells (1×10⁷ cells/mouse). No exogenouscytokines were injected into the mice. A, tumor growth measured as lightemission in a representative cohort of 5 mice from each group of NT andCAR-GD2 T cell-treated animals. B, survival curve of mice engrafted withthe P1143 (95% GD2 positive) tumor cells receiving either tumor alone,NT T cells or CAR-GD2 T lymphocytes.

FIG. 7 shows cytolytic activity of T-lymphocytes redirected to expressGD2-CAR against normal cell lines. The inventors used a ⁵¹CR releaseassay to evaluate the cytotoxic activity of T lymphocytes expressingCAR-GD2. Target cells were allogeneic peripheral blood mononuclear cells(PBMCs) and GD2-skin fibroblasts. The inventors also used a GD2+mesenchymal stem cell (MSC) line, which was susceptible to CAR-GD2 Tcell killing. Data illustrate the mean and SD of ⁵¹Cr release from 4 Tcell lines after 6 and 18 hours incubation. Non-transduced T cells didnot kill any of the targets tested.

FIG. 8 shows T lymphocytes expressing CAR-GD2 and co-expressingCD28-OX40 endodomains proliferate in response to GD2+ melanoma celllines. CAR-GD2 T cells labeled with CFSE, to evaluate T cell division,were co-cultured (Ratio T cells:tumor cells of 20:1) with three melanomacell lines expressing dim (CLB), intermediate (SENMA) or high (P1143)levels of GD2. Non transuced (NT) T lymphocytes were used as negativecontrols. CFSE expression by T cells was analyzed by FACS on day 4. OnlyT cells expressing CAR-GD2 divided multiply in response to GD2+ melanomacell lines. Data are representative of repeat experiments.

FIGS. 9A and 9B show GD2 expression in lung cancer. FIG. 9A showsexpression in small cell lung cancer. FIG. 9B shows expression innon-small cell lung cancer.

FIG. 10 shows flow cytometry for both small cell and non small cell lungcancer.

FIG. 11 shows transduction rates with GD2 CAR using GD2 specificproteins.

FIGS. 12A and 12B demonstrate that GD2 CAR-transduced T lymphocytesrecognize and kill lung cancer cell lines. FIG. 12A shows killing ofsmall cell lung cancer, and FIG. 12B shows killing of non-small celllung cancer.

FIG. 13 demonstrates that GD2 CAR-transduced T lymphocytes secreteimmunostimulatory cytokines in coculture of GD2 positive lung cancercell lines.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, certain details are set forth such asspecific quantities, sizes, etc. so as to provide a thoroughunderstanding of the present embodiments disclosed herein. However, itwill be obvious to those skilled in the art that the present disclosuremay be practiced without such specific details. In many cases, detailsconcerning such considerations and the like have been omitted inasmuchas such details are not necessary to obtain a complete understanding ofthe present disclosure and are within the skills of persons of ordinaryskill in the relevant art.

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

I. DEFINITIONS

As used herein, the term “costimulatory molecule” refers to a molecularcomponent that promotes activation, proliferation and effector functionof a T cell after engagement of an antigen specific receptor.

As used herein, the term “cytoplasmic signaling domain” refers to thecomponent of a co-stimulatory molecule or cytokine receptor that existsinside the cell and is responsible for transducing the external signalreceived to the internal metabolic processes of the cell, therebyaltering its phenotype and function.

In embodiments of the present invention, the overexpression of GD2 byhuman primary melanoma cells allows these cells to be targeted in vitroand in vivo by GD2 CAR-expressing primary T cells, and incorporation ofendodomains from both CD28 and OX40 molecules (Pule et al., 2005)mediates costimulation of the T lymphocytes, inducing T cell activation,proliferation, and cytotoxicity against GD2-positive melanoma cells.

In particular embodiments of the invention, there are methods forkilling metastatic melanoma cells using genetically manipulated T-cellsthat express a chimeric antigen receptor (CAR) directed against theganglioside antigen GD2. Engagement (antigen binding) of this CAR leadsto activation of the linked T-cell receptor ζ chain and thecostimulatory molecules CD28 and OX40. The present invention leads tothe suppression of metastatic melanoma xenografts in vivo, and theskilled artisan recognizes that such practices extrapolate to non smallcell lung cancer, in certain embodiments.

II. SCFV ANTIBODIES

In particular embodiments of the invention, the CAR receptor comprises asingle-chain variable fragment (scFv) that recognizes GD2. The skilledartisan recognizes that scFv is a fusion protein of the variable regionsof the heavy (VH) and light chains (VL) of immunoglobulins, connectedwith a short linker peptide of ten to about 25 amino acids. The linkermay be rich in glycine for flexibility and/or it may have serine orthreonine for solubility, in certain cases. In a particular embodiment,the 14g2a scFv antibody is used in the CAR. The scFv may be generated bymethods known in the art.

Other examples of ScFv made and successfully tested in pre-clinicalstudies include, but are not limited to, CD20, CD19, CD30, Her2, kappalight chain, and lambda light chain, and in certain embodiments one ormore of these are employed in the invention.

In certain aspects, one can use cytokine exodomains or otherligand/receptor molecules as exodomains to provide targeting to thetumor cells.

III. COSTIMULATORY MOLECULES

The skilled artisan recognizes that T cells utilize co-stimulatorysignals that are antigen non-specific to become fully activated. Inparticular cases they are provided by the interaction betweenco-stimulatory molecules expressed on the membrane of APC and the Tcell. In specific embodiments, the one or more costimulatory moleculesin the chimeric receptor come from the B7/CD28 family, TNF superfamily,or the signaling lymphocyte activation molecule (SLAM) family. Exemplarycostimulatory molecules include one or more of the following: B7-1/CD80;CD28; B7-2/CD86; CTLA-4; B7-H1/PD-L1; ICOS; B7-H2; PD-1; B7-H3; PD-L2;B7-H4; PDCD6; BTLA; 4-1BB/TNFRSF9/CD137; CD40 Ligand/TNFSF5; 4-1BBLigand/TNFSF9; GITR/TNFRSF18; BAFF/BLyS/TNFSF13B; GITR Ligand/TNFSF18;BAFF R/TNFRSF13C; HVEM/TNFRSF14; CD27/TNFRSF7; LIGHT/TNFSF14; CD27Ligand/TNFSF7; OX40/TNFRSF4; CD30/TNFRSF8; OX40 Ligand/TNFSF4; CD30Ligand/TNFSF8; TACI/TNFRSF13B; CD40/TNFRSF5; 2B4/CD244/SLAMF4;CD84/SLAMF5; BLAME/SLAMF8; CD229/SLAMF3; CD2 CRACC/SLAMF7;CD2F-10/SLAMF9; NTB-A/SLAMF6; CD48/SLAMF2; SLAM/CD150; CD58/LFA-3; CD2;Ikaros; CD53; Integrin alpha 4/CD49d; CD82/Kai-1; Integrin alpha 4 beta1; CD90/Thy1; Integrin alpha 4 beta 7/LPAM-1; CD96; LAG-3; CD160;LMIR1/CD300A; CRTAM; TCL1A; DAP12; TIM-1/KIM-1/HAVCR; Dectin-1/CLEC7A;TIM-4; DPPIV/CD26; TSLP; EphB6; TSLP R; and HLA-DR.

The CAR of the invention may employ one, two, three, four, or morecostimulatory molecules.

IV. EFFECTOR DOMAIN OF THE T-CELL RECEPTOR ZETA CHAIN

The effector domain is a signalling domain that tranduces the event ofreceptor ligand binding to an intracellular signal that partiallyactivates the T lymphocyte. Absent appropriate co-stimulatory signals,this event is insufficient for useful T cell activation andproliferation.

V. COMBINATION THERAPY

In certain embodiments of the invention, methods of the presentinvention for clinical aspects are combined with other agents effectivein the treatment of hyperproliferative disease, such as anti-canceragents. An “anti-cancer” agent is capable of negatively affecting cancerin a subject, for example, by killing cancer cells, inducing apoptosisin cancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cancer cells withthe expression construct and the agent(s) or multiple factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver, et al., 1992). In the context ofthe present invention, it is contemplated that cell therapy could beused similarly in conjunction with chemotherapeutic, radiotherapeutic,or immunotherapeutic intervention, in addition to other pro-apoptotic orcell cycle regulating agents.

Alternatively, the present inventive therapy may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and present invention are appliedseparately to the individual, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and inventive therapy would still be ableto exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4,5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, present invention is “A” and thesecondary agent, such as radio- or chemotherapy, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the inventivecell therapy.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, abraxane, altretamine, docetaxel, herceptin,methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

In specific embodiments, chemotherapy for melanoma is employed inconjunction with the invention, for example before, during and/or afteradministration of the invention. Exemplary chemotherapeutic agents formelanoma include at least dacarbazine (also termed DTIC), temozolimide,paclitaxel, cisplatin, carmustine, fotemustine, vindesine, vincristine,or bleomycin.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

Immunotherapeutics generally rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy could thus be used as part of a combined therapy, inconjunction with the present cell therapy. The general approach forcombined therapy is discussed below. Generally, the tumor cell must bearsome marker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155.

Immunotherapy for melanoma may include interleukin-2 (IL-2) orinterferon (IFN), for example.

D. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the present invention clinical embodiments. A varietyof expression products are encompassed within the invention, includinginducers of cellular proliferation, inhibitors of cellularproliferation, or regulators of programmed cell death.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

F. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abililties of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Example 1 Exemplary Materials and Methods

Establishment of cell lines. After informed consent, tumor biopsies(from metastatic skin lesions) were obtained from five patients withstage III or IV melanoma. The tumor tissue was minced and the fragmentsresuspended in 30 mL of digestion medium containing DNAse at 30 U/mL,hyaluronidase at 0.1 mg/mL, and collagenase at 1 mg/mL (all fromSigma-Aldrich), in complete medium prepared as follows: DMEM (Cambrex)supplemented with 10% of heat inactivated FCS (HyClone), 200 IU/mLpenicillin, 200 mg/mL streptomycin, 100 mg/mL gentamicin (Invitrogen),and 2 mmol/L GlutaMAX (Invitrogen). After 4 h incubation at 37° C. in 5%CO₂, the cell suspension supernatant (free of tissue debris) wascollected, transferred to a new tube, and then centrifuged at 400×g for5 min. Cells were resuspended in a 6-well plate in fresh complete mediumcontaining 1 mmol/L sodium pyruvate (Invitrogen), and cultured at 37° C.in 5% CO₂. Culture medium was renewed every 72 h. At day 6, theantibiotics present in the complete medium were reduced to 100 IU/mLpenicillin and 100 mg/mL streptomycin.

When tumor cells reached confluence, they were transferred to a T25flask for further amplification. The established tumor cell lines (CLB,SENMA, Plaode, RR-371953, and P1143) were characterized by fluorescenceactivated cell sorting (FACS) analysis (MCSP and GD2) andimmunofluorescence (gp100, MAGE-1, and MART-1). Low passage number (<20)of the primary melanoma cell lines was used in in vitro and in vivoexperiments.

Normal mesenchymal stem cells and normal skin fibroblasts were generatedin laboratory as previously described (Yvon et al., 2003; Gottschalk etal., 2003), and the K562 cell line was obtained from the American TypeCulture Collection. All cell lines were maintained in RPMI (Hyclone)supplemented with 10% heat inactivated FCS, 100 IU/mL penicillin, 100mg/mL streptomycin, 1 mmol/L sodium pyruvate (Invitrogen), and 2 mmol/LGlutaMAX. Six established melanoma cell lines, isolated from surgicalspecimens at Istituto Nazionale Tumori, Milan, were also used to screenGD2 expression.

Mononuclear cells. Peripheral blood, obtained after informed consentfrom normal donors, was processed over Ficoll gradients, and theresulting peripheral blood mononuclear cells (PBMC) were cultured incomplete T-cell medium containing 45% RPMI and 45% Click's mediumsupplemented with 10% heat inactivated FCS, 100 IU/mL penicillin, 100mg/mL streptomycin, and 2 mmol/L GlutaMAX.

Retroviral constructs. The 14g2a scFv sequence was cloned in the SFGretroviral backbone in frame with the human IgG1-CH2CH3 domain, followedby the CD28 and OX40 endodomains and the ζ-chain of the T-cellreceptor/CD3 complex, to form the 14g2a-CD28-OX40-ζ (CAR-GD2) constructas previously described (Pule et al., 2005). Vectors encoding theFirefly Luciferase gene (FF-Luc.) or the eGFP protein were also used totrack cell survival and proliferation in vivo, as previously described(Savoldo et al., 2000). The RD114 retrovirus envelope (RDF plasmid) andthe MoMLV gag-pol (PegPam3-e plasmid) were used to engineer theretroviral vectors.

Retrovirus production and transduction. Transient retroviralsupernatants were produced by cotransfection of 293T cells with thePegPam-e, RDF, and the desired SFG vectors (CAR-GD2, eGFP, or FF-Luc)using the Fugene6 transfection reagent (Roche), and used to transduceOKT3 (Ortho Biotech) activated PBMCs, as previously described (Vera etal., 2006).

The 4405M, CLB, SENMA, and P1143 melanoma cell lines were transfectedwith retroviral vectors encoding either eGFP or FF-Luc. The inventorsplated 1×10⁵ tumor cells in 1 well of a 6-well plate and the cells weregrown to 60% to 70% confluency. Culture medium was replaced by theappropriate retroviral supernatant (1.5 mL/well), and 1 μg of polybrenewas added. When the tumor cells reached confluency, they weretrypsinized and plated in a T25 flask. The FF-Luc-transduced cells werethen selected with puromycin (Sigma-Aldrich) at 1 μg/mL. TheeGFPtransduced tumor cell lines did not require selection as >98% of thecells were eGFP-positive postretroviral transduction.

Flow cytometry. FITC-, phycoerythrin (PE)-, or periodin chlorophyllprotein (perCP)-conjugated anti-CD4, -CD8, -CD80 and -CD86 monoclonalantibodies (all from Becton-Dickinson) were used to label lymphocytes,whereas anti-MCSP-PE (Miltenyi-Biotech Inc.) and a purified anti-GD2monoclonal antibody (Becton-Dickinson Pharmingen) were used to stain themelanoma cells. A secondary antibody (RAM-IgG2a+b-PE; Becton-Dickinson)was added to detect the anti-GD2 (IgG2a) antibody by indirectimmunofluorescence. CAR expression by transduced T lymphocytes wasdetected using a monoclonal anti-idiotype, 1A7 (TriGem, Titan), followedby staining with the secondary antibody RAM-IgG1-PE (Becton-Dickinson;Rossig et al., 2001). The proliferation of nontransduced and transducedT cells, in the presence or absence of tumor cells, was evaluated byFACS analysis after labeling T cells with CFSE (Invitrogen) according tothe manufacturer's instructions.

Cytotoxicity assays. The cytotoxic activity of the nontransduced andCAR-GD2 T lymphocytes was evaluated in a standard ⁵¹Cr release assay, aspreviously described (Pule et al., 2005; Vera et al., 2006). Isotoperelease was evaluated at 6 and 18 h in cultures with effector-to-target(E:T) ratios of 40:1, 20:1, 10:1, and 5:1, using a gamma counter(Perkin-Elmer).

Coculture experiments. Seven days after transduction, nontransduced andCAR-GD2 cells were collected, counted, and plated at 5×10⁵ cells/well ina 24-well plate at 20:1 ratio with eGFP-expressing (>98%+) tumor cells.Cytokine release after 24 h of culture was measured using the CBAarray(BD Bioscience) and the percent of CD3-positive T cells andeGFP-positive tumor cells was evaluated by FACS analysis at day 5 ofcoculture, after treatment with 0.5% trypsin EDTA (Invitrogen) to detachadherent cells.

Xenogeneic SCIDmo use model of melanoma. To assess the in vivo antitumoractivity of the CAR-GD2 T lymphocytes, an exemplary SCID mouse model wasused and the P1143 or 4405 M melanoma line expressing FF-Luciferase.SCID mice (8 to 9 weeks old) were sublethally irradiated (250 rad) andinjected i.v. with 2×10⁶ tumor cells. Tumor cell engraftment wasmonitored using the IVIS 100 imaging system (Caliper Lifesciences), andon days 4 and 21, 1×10⁷ nontransduced or CAR-GD2 T lymphocytes wereinjected i.v. The animals were imaged weekly to evaluate tumor growth,and photon emission from luciferase-expressing cells was quantifiedusing the “Living Image” software provided with the IVIS system (CaliperLifesciences). Briefly, after drawing a region of interest over thetumor region, the intensity of the signal measured was expressed astotal photons/s/cm2 (p/s/cm²/sr).

Statistical analysis. For cytotoxicity and cytokine production, resultswere presented as mean±SD and paired Student's t test was used todetermine statistical significance. For the bioluminescence results, thesignal intensity was log-transformed and summarized using mean±SD atbaseline and multiple subsequent time points for each group of mice.Changes in intensity of signal from baseline at each time point werecalculated and compared using paired t-tests or Wilcoxon signed-rankstest. P<0.05 was considered statistically significant.

Example 2 Expression of GD2 by Primary Melanoma Cells

To characterize GD2 as a target for CAR-directed T cell therapy, primarymelanoma cells were dissociated from five patients after biopsy ofcutaneous metastatic melanoma and an additional six established celllines were used in a study. Cells from the five patients and four of theestablished lines expressed GD2 on immunofluorescence staining, andbetween 17% and 95% of the cells were positive, with variable intensityof expression (FIG. 1). Expression of GD2 was used on a normal skinfibroblast cell line and it was confirmed that it did not express theganglioside. To confirm the absence of nonneoplastic cells in theprimary culture, expression of MCSP on the cells (Table 1) was examined,and to compare the frequency of expression of GD2 with that of otherknown melanoma tumor associated antigens, theexpression of gp100, MART1,and MAGE-1 was also measured. The percentage range of GD2-positive cellswas comparable with the range of malignant cells expressing these threeother melanoma-associated antigen cells (Table 1). All cell populationsstudied were negative for expression of the costimulatory molecules CD80or CD86.

TABLE 1 Characterization of the melanoma cell lines. Expression of GD2,MCSP, gp100, MART1 and Mage-1 is presented. MCSP and GD2 expression wasevaluated by FACS analysis and fluorescence used to determine theexpression of gp100, MART1 and Mage-1. GD2 MCSP gp100 MART1 Mage-1(%/MFI) (%) (%) (%) (%) CLB 19/182 100 99 11 46 SENMA 45/884 100 92 8098 PLAODE 22/219 100 11 22 3 RR-371952 17/775 100 47 99 1 P1143 95/83810 100 92 10 18588M 85/162 100 1 3 80 18732M 93/150 100 99 76 26 4405M 0/NA 100 26 9 1 10538P 80/142 100 57 14 62 879M  0/NA 100 59 71 558959M 77/71 100 99 32 82

T cells expressing a GD2-specific chimeric receptor kill GD2-positivemelanoma cell lines. T cells from four healthy donors were transducedwith a vector encoding the 14g2a single-chain antibody linked to ζ andto the endodomains of the two costimulatory molecules CD28 and OX40,which enhance the activation, proliferation, and cytotoxicity of T cellsproduced by the CAR after antigen engagement (Pule et al., 2005). Fivedays after transduction, the expression of GD2-specific CAR was measuredby flow cytometry using the anti-14g2a idiotypic antibody 1A7, and itwas found that 95% of cells transduced with the 14g2a-CD28-OX40-ζretroviral vector were CAR positive (range, 93-97%; FIG. 2A). TheCAR-GD2 construct transduced CD4-positve and CD8-positive T cellpopulations with equal efficiency (FIG. 2B).

To mimic the range of GD2 expression seen on primary melanoma cells, theability was measured of CAR-GD2 T cells to kill three melanoma celllines with different GD2 expression. P1143 was a high expressor [95%positive, mean fluorescence intensity (MFI)=838]; SENMA was intermediate(45% positive, MFI=884); CLB was low (19% positive, MFI=182), andfinally, the melanoma cell line 4405 M was used as a GD2-negative tumorcell control. At 6 hours and 18 hours, ⁵¹Cr release assays showed thatthe antitumor activity was proportional to the level of GD2 antigenexpression (FIG. 3). As anticipated, the CAR-GD2 T cells had littleactivity against the GD2-negative tumor cell line (4405 M), theGD2-NK-cell target line K562, or against normal skin fibroblasts orPBMCs that are also GD2 negative (FIG. 7).

CAR-GD2 T cells were, however, able to kill a mesenchymal stem cell linepositive for GD2 (95% positive, MFI=799; FIG. 7). Of note, suchcross-reactivity with GD2-positive normal mesenchymal stem cells has notproduced discernible adverse effects in any clinical trial of GD2monoclonal antibodies in patients with neuroectodermal tumors (Saleh etal., 1995; Murray et al., 1995) or in a phase I study of CAR-GD2 T cellsin patients with neuroblastoma (Pule et al., 2008).

CAR-GD2-expressing T cells secrete cytokines upon stimulation withGD2-expressing melanoma cells. Functional activation ofCAR-GD2-expressing T cells following their exposure to GD2-positivemelanoma cells was measured by cytokine release assay. As describedabove, GD2-positive target cells were killed by T cells expressing theCAR, and significant interleukin 2 (IL-2), IL-5, IFN-γ, and tumornecrosis factor α (TNF-α) release occurred during coculture with thethree melanoma lines.

As FIG. 4 shows, the quantity of IL-2, IL-5, IFN-γ, and TNF-α secretedby the CAR-GD2 T cells after 24 hours of culture correlated with thelevel of GD2 expression on the target cells, and was highest for P1143and lowest for CLB. Neither IL-4 nor IL-10 was detected in thesupernatants of stimulated cells.

CAR-GD2 induces sustained killing and clonal expansion in cocultureexperiments. It was next determined whether the killing and cytokinerelease mediated by CAR-GD2 T cells could lead to CAR-T cellproliferation and tumor cell eradication in vitro in a 5-day cocultureexperiment. CFSE-labeled control or CAR-GD2 T cells were used todetermine whether CAR stimulation by CAR-GD2-expressing T cells induceseffector T cell proliferation.

Nontransduced T cells proliferated only in the presence of exogenousIL-2 (100 U/mL), whereas proliferation of CAR-expressing T cellsincreased in response to all three GD2-expressing tumor cell lines,irrespective of whether these tumor cells expressed high, intermediate,or low levels of GD2 (FIG. 8). To discover if these expanded CAR-GD2 Tcells were functional, tumor cells were labeled with eGFP and werecocultured at the CAR-T cell:tumor cell ratio of 20:1 in the absence ofIL-2. After 5 days of culture, viable GFP-positive cells were enumeratedby flow cytometric analysis. FIG. 5 shows that viable tumor cells wereeradicated in cocultures with T cells expressing CARGD2 but not incocultures with nontransduced T cells. Hence CAR-GD2 T cells proliferatein vitro in response to the GD2 antigen and eradicate melanoma cellsthat express the antigen. As expected, the GD2-negative cell line 4405 Mwas not killed in the 5-day coculture experiment, showing GD2 antigenrecognition is essential for the activity of CAR-GD2-expressing T cells.

Adoptive transfer of GD2-specific T cells provide antitumor effect in axenogeneic SCIDmodel. The antitumor activity was measured of CAR-GD2 Tcells in vivo. To monitor tumor cells in vivo, the firefly luciferase(FFLuc) gene was expressed in 4405 M and P1143 cells, together with thepuromycinresistance gene. After puromycin selection, 2×10⁶ FFLuc-P1143or 4405 M tumor cells were injected i.v. into SCID mice.

After 4 days, FFLuc expression was evaluated by bioluminescence imagingand the mice were divided into three groups that received nontransducedT cells or T cells expressing CAR-GD2 at 1×10⁷ i.v. and finally a groupthat received tumor cells alone. A second injection of nontransduced orCAR-GD2 T cells was given at day 21 and luciferase signal was measuredevery week in the 10 mice of each of the groups. FIG. 6A shows fiverepresentative mice from the nontransduced and CAR-GD2 T cells group,and shows that tumor grew rapidly in the lungs of mice receivingnontransduced T cells. By contrast, the tumors in mice receiving T cellsexpressing CAR-GD2 diminished within 48 to 72 hours of injection, andluciferase derived remained largely absent in the group receiving CARGD2T cells. Although the survival of the mice receiving the tumor cellsalone or tumor cells plus nontransduced T cells was 68±6 days and 72±12days, respectively (P=0.03), 80% of the mice from the group receivingCAR-GD2 T cells were still alive at day 100 and showed a significantsurvival advantage when receiving CAR-GD2-specific T cells (P=0.006;FIG. 6B). Finally, no tumor regression was reserved when CAR-GD2 T cellswere infused in mice bearing GD2-4405 M tumor cells (FIG. 6A).

Example 3 Significance of Certain Embodiments of the Invention

In particular aspects of the invention, the ganglioside antigen GD2 isexpressed on the majority of primary melanoma cell lines, and T cellsengineered to express a CAR directed to this antigen are able torecognize and lyse GD2-positive melanoma target cells in vitro and in aSCID mouse model in vivo. The transgenic receptor construct included thesignaling endodomains of the CD28 and OX40 costimulatory molecules, andredirected T cells showed activation, proliferation, and cytokinerelease after T-cell receptor engagement by GD2.

Melanoma has long been a target of cellular immunotherapies directed tothe tumor-associated antigens expressed by the malignant cells. Althoughearlier clinical research focused on reinfusion of expanded tumorinfiltrating lymphocytes, efforts have recently been directed againstthe cancer testis series of antigens such as MAGE and the melanocytedifferentiation proteins such as MART-1, by generation of T cellsexpressing conventional αβ-T-cell receptors specific for these antigens.These receptors recognize peptide fragments in association with MHCclass I molecules, and are therefore restricted in their patient rangeto individuals with the appropriate MHC polymorphism. Moreover, they areunable to recognize tumor subclones in which the antigen processingmachinery is deficient. T cells that express synthetic or chimericreceptors that recognize unprocessed structures on the cell surface maythus have an advantage over T cells whose tumor reactivity is mediatedthrough their native receptor.

It has been known for some time that the ganglioside GD2 is expressed bytumors derived from neuroectoderm, including neuroblastoma, sarcoma, andsmall lung cancer. This tumor-associated carbohydrate antigen is alsoexpressed by many melanoma cells (Cheresh et al., 1984), in which it isinvolved in cell adhesion and may contribute to metastasis (Hakomori,2001). Although GD2 is present on the surface of many melanoma cells, itis absent on most normal tissue, with only limited expression in brainand on peripheral nerves, making this ganglioside an attractive targetfor adoptive cell therapy in metastatic melanoma (Hersey et al., 1998).

GD2 monoclonal antibodies have already been used with benefit inpatients with other GD2-positive malignancies, such as neuroblastoma,but melanoma cells have more variable (and usually lower) expression ofthe antigen. Hence, the benefits of GD2 antibody infusion in melanomahave been limited (Saleh et al., 1992; Murray et al., 1994). The levelof GD2 expression on melanoma cells, however, is evidently sufficient toproduce a cytotoxic response from T cells expressing the same monoclonalantibody binding site in the form of a chimeric T-cell receptor. Therewas complete killing of the tumor cells even when GD2 expression waslow, consistent with previous observations that even tumor cells withdim expression of the targeted antigen can be eliminated by CAR-modifiedT cells (Vera et al., 2006). The killing of cells that are resistant toantibodies of the same specificity may be related to the improvedavidity of multiple antibody-derived binding domains when they arearrayed on a cell surface rather than existing as bivalent molecules insolution, or it may reflect a superior cytolytic activity of T effectorcells compared with antibody (Weijtens et al., 2007).

Hence, tumor cells with dim antigen expression can be completelyeliminated after coculture experiments, even when short term assaysbased on ⁵¹Cr release assay may produce lower immediate cytotoxicitythan tumor cells with high antigen expression Like most malignancies,melanoma cells lack expression of the T-cell costimulatory moleculesrequired for complete activation of T lymphocytes that engagetumor-associated antigens through their native or chimeric receptors.Hence, to optimize T cell triggering and effector function, the chimericreceptor was coupled to co-stimulatory endodomains to increase T cellsurvival and expansion. Following CAR engagement, endodomains fromsingle co-stimulatory molecules, such as CD28, 4-1BB, or OX40, into theCAR may be sufficient to activate the cellular components of the killingmachinery and to produce IL-2 release and T cell proliferation(Willemsen et al., 2005; Imai et al., 2004). It has been previouslyshown, however, that the simultaneous expression in cis of twoendodomains such as CD28 and OX40 within a GD2-CAR produces superior Tcell proliferation and effector function than expression of a singlecostimulatory endodomain (Pule et al., 2005). In certain embodiments,this benefit occurs because both CD28 and OX40 signals are bothfunctional, and produce greater activation of NF-κB than eitherendodomain alone, because they act through two independent pathways(Pule et al., 2005).

If adoptive transfer of CAR-modified T cells in melanoma is to be ofclinical value, it will be essential to be able to treat metastaticdisease. Accordingly, the effects of the CAR-GD2 T cells were studied inan exemplary xenograft lung metastatic model. Human T cells expressingthe 14g2a-CD28-OX40-ζ CAR produced significant antitumor activity inthis model, but were unable to completely eradicate the disease. Thisincomplete benefit may reflect the difficulties of sustaining human Tcell function and trafficking in a xenogeneic environment, or it mayalso represent the limitations of even the combination of CD28 and OX40endodomains, which on their own cannot completely recapitulate thetemporo-spatial features of the costimulatory events required to sustainT cell activation physiologically (Pule et al., 2008; Heemskerk et al.,2004).

Thus, in aspects of the invention, GD2 on melanoma cells is a usefultarget for CAR-T cells. Administration of such cells will usefullycomplement other cellular immunotherapies and biotherapies for thisdisease, in at least some embodiments.

Example 4 Targeting the Disialo-Ganglioside GD2 in Lung Cancer UsingChimeric Antigen Receptor (CAR) T Lymphocytes

FIGS. 9A-9B demonstrate GD2 expression in lung cancer: To study theexpression of GD in lung cancer cell the inventors performedcytochemistry on a number of lung cancer cytospin specimens and flowcytometry. FIG. 9A shows expression in small cell lung cancer, and FIG.9B shows expression in non-small cell lung cancer.

These findings were validated using flow cytometry for both small celllung cancer and non-small cell lung cancer (FIG. 10).

Transduction of T lymphocytes was employed to express GD2 CD28.zeta CAR.The inventors used a retroviral system to transduce human peripheralblood lymphocytes to express the GD2-specific third generation CARmolecule with a CD28, OX40 and zeta signaling domains (FIG. 11). Morethan 50% transduction rates was consistently obtained with variousdonors.

In FIGS. 12A-12B, GD2 CAR-transduced T lymphocytes recognize and killlung cancer cell lines. The inventors used standard 4-6 hour ⁵¹Crrelease assays to assess the degree of cytolysis of exemplary lungcancer cell lines at various tumor to T cell ratios. The neuroblastomacell line LAN1 was used as a positive control. Lymphoblastoid cell lines(LCL) were used as the negative control. FIG. 12A shows killing of smallcell lung cancer, and FIG. 12B shows killing of non-small cell lungcancer.

GD2 CAR-transduced T lymphocytes secrete immunostimulatory cytokines incoculture of GD2 positive lung cancer cell lines (FIG. 13). ELISA wasperformed to detect the cytokine release (IFNγ and IL-2) 24 to 48 hoursafter co-culture initiation. GD2 expressing T cells secretedimmunostimulatory cytokines upon encounter of GD2 positive lung cancercells above non-transduced controls from the same donor.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein their entirety.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. A method of targeting a cancer cell having a GD2antigen, comprising the steps of providing to the cell a cytotoxic Tlymphocyte with a chimeric receptor that recognizes the GD2 antigen. 2.The method of claim 1, wherein the cancer cell is in vitro or in vivo.3. The method of claim 1, wherein the chimeric receptor comprisesantibody that binds GD2
 4. The method of claim 2, wherein the antibodyis a scFv antibody.
 5. The method of claim 2, wherein the antibody isthe 14g2a scFv antibody.
 6. The method of claim 1, wherein the chimericreceptor comprises the effector domain of the T-cell receptor zeta chainor related signal transduction endodomains derived from the T cellreceptor.
 7. The method of claim 1, wherein the chimeric receptorcomprises one or more costimulatory molecules.
 8. The method of claim 6,wherein the costimulatory molecules comprise CD28, OX40, 4-1BB, or acombination thereof.